The field of the invention is the use of xcex3-phosphoester nucleoside triphosphates in polymerase reactions.
The human immunodeficiency virus infects over 40 million people worldwide and the Hepatitis C virus has infected approximately 2% of the world""s population. The economic and medical impacts of such emerging epidemics demonstrate clearly the need to rapidly and effectively assess the efficacy of viral inhibitors. Assaying inhibitors of viral RNA or DNA synthesis is a time consuming and costly process that often requires radioisotopes. This screening process is expensive, time consuming, and requires special handling due to the use of radioisotopes. Previous attempts to make colorimetric or fluorescent nucleotide analogs useful for the detection of polymerase activity primarily employ a detectable chromophore or fluorophore attached to the base or ribose portion of a nucleotide. The signal is then incorporated into the newly formed product nucleic acid, hence necessitating an often lengthy and labor-intensive step to separate the products from the reactants.
Several companies sell products that incorporate a detectable reagent into the product of polymerase synthesis, including Boehringer (Genius kit), Life Technologies INC., GIBC/BRL, Sigma (biotinylated nucleotides, fluorescent nucleotides), Molecular Probes Inc. (a large range of fluorescent and caged nucleotides), Li-Cor (dyes attached to DNAs for DNA sequencing), etc. Reports of xcex3-phosphoesters of nucleoside triphosphates have described them as non-hydrolyzable and used them in solid phase affinity purification protocols, e.g. Clare M. M. Haystead, et al., Gamma-phosphate-linked ATP-Sepharosefor the affinity purification of protein kinases, Eur. J. Biochem. 214, 459-467 (1993), esp. p.460, col. 2, line 23. We synthesized large numbers of xcex3-phosphoester nucleoside triphosphates and found that while they are indeed non-hydrolyzable by many enzymes, they are often suitable substrates for RNA and DNA polymerases.
The invention provides methods and compositions for polymerizing a particular nucleotide with a polymerase. In general, the method involves (a) forming a mixture of a polymerase and a nucleoside triphosphate (NTP) comprising xcex1, xcex2 and xcex3 phosphates and a xcex3-phosphate phosphoester-linked functional group; and (b) incubating the mixture under conditions wherein the polymerase catalyzes cleavage of the NTP between the xcex1 and xcex2 phosphates, liberating a pyrophosphate comprising the functional group and polymerizing the resultant nucleoside monophosphate. i.e. incorporates the nucleoside monophosphate in a nascent polynucleotide.
A variety of functional groups compatible with the polymerization reaction are provided. In one embodiment, the functional group is a detectable label and the method further comprises the step of detecting the label, wherein a wide variety of chromogenic and luminogenic labels are provided.
In another embodiment, the functional group is a cell delivery enhancing moiety, xe2x80x94OR, wherein R is independently selected from: substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl, each inclusive of carbocyclic and heterocyclic. These substituents provide enhanced therapeutic availability through enhanced gut or blood stability, cellular and/or membrane permeability, host phosphatase stability, etc. This aspect provides a wide variety of generally membrane permeable, relatively hydrophobic R substituents.
In another embodiment, the functional group is a polymerase specificity enhancing moiety, xe2x80x94OR, wherein R is independently selected from: substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl, each inclusive of carbocyclic and heterocyclic. These substituents are readily identified in comparative and competitive enzyme assays.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation.
The general method involves forming a mixture of a polymerase and a nucleoside triphosphate (NTP) comprising xcex1, xcex2 and xcex3 phosphates and a xcex3-phosphate phosphoester-linked functional group; and incubating the mixture under conditions wherein the polymerase catalyzes cleavage of the NTP between the xcex1 and xcex2 phosphates, liberating a pyrophosphate comprising the functional group and polymerizing the resultant nucleoside monophosphate. The mixture generally also comprises a template, a nascent polynucleotide and other reagents which facilitate the polymerase reaction, such as salts, buffers, etc. The mixture may be formed in any context, such as in vitro, within a virus or cell, etc. Monitoring polynucleotide synthesis by continuous measurement assays can be performed with homopolymeric templates and a single labeled NTP. Alternatively, continuous monitoring of polymerase activity can be performed by synthesizing all four modified nucleotides, rendering all nucleotides resistant to alkaline phosphatase.
A wide variety of polymerases may be employed, including DNA- and/or RNA-dependent RNA polymerases and DNA- and/or RNA-dependent DNA polymerases. Depending on the application, the polymerase may reside in a cell or virus, such as within its natural host cell environment, or be isolated or in vitro, such as isolated from cellular, microbial and/or viral source material. In many cases, suitable polymerases are commercially available, e.g. Taq, a DNA-dependent DNA polymerase (Boehringer Mannheim (BM) catalog #1-146-165); Klenow fragment (BM catalog #1-008-404) reverse transcriptase (RT), e.g. Moloney murine leukemia virus RT (BM catalog #1-062-603), human immunodeficiency virus RT (BM catalog #1-465-333). Exemplary targetable pathogenic polymerases include reverse transcriptases (e.g. from HIV, and hepatitis B), viral RNA polymerases (e.g. from HCV and Dengues virus) and DNA polymerases (e.g. Herpes and Epstein-Barr virus DNA polymerases).
A wide variety of NTPs which function as substrates for the targeted polymerase may be used in the method. The nucleotide may comprise a conventional purine or pyrimidine base, such as adenine, guanine, cytosine, uracil and thymine, which may be substituted with a variety of known modifications, such as methyl, amine, halide (e.g. 5-fluorouracil), etc., and a pentose (including ribose and deoxyribose), which may also be substituted with a variety of known modifications, such as amine, o-methyl ester, 2xe2x80x2-deoxy, etc. The nucleotide may also comprise a nucleotide analog which functions as a substrate of the target polymerase, such as acyclovir, gangcyclovir, zidovudine (AZT), etc.
The NTP comprises one or two xcex3-phosphate phosphoester-linked functional groups (i.e. mono phosphoester or phosphodiesters), providing a functionality such as enhancement of reaction product detectability, cell delivery, polymerase specificity, etc. and/or a reaction product functionality such as a therapeutic or protherapeutic, which functionalities may be provided by a wide variety of structural moieties. Numerous exemplary suitable functional groups are disclosed herein and/or readily identified in convenient screens, such as the polymerase, targeting and specificity screens described below.
In one embodiment, the functional group is a detectable label such as a chromogenic or luminogenic (including fluorogenic) label. In this embodiment, the method generally further comprises, after the incubating step, the step of detecting the label. Accordingly, this aspect of the invention provides safe, simple, efficient, nonradioactive, quantitative assays to detect nucleic acid (RNA and DNA) synthesis by polymerases. The methods need not require a separation step where the substrate does not absorb at the detection wavelength until after it is used in a polymerase reaction. Detection can be effected with conventional spectrophoto/fluori-meters routinely used in research laboratories and classrooms. The methods provide real-time colorimetric assays that easily and efficiently detect and quantify DNA and RNA synthesis.
Chromo/luminogenic NTP analogs offer many advantages to traditional assays that use radioisotopes. They are inexpensive to produce, are stable in the presence of plasma, can be detected with high sensitivity, with a multi-fold signal to background ratio, e.g. at least 7-fold. Furthermore, the assay can be monitored continuously and will give clearly distinguishable results in short time-frames, e.g. within 15 minutes. These assays are readily adapted to a 96-well plate format and performed in commercial ELISA plate readers.
The colorimetric nucleotide analogs are easily detected with normal laboratory equipment. The ability to have individual nucleotides tagged with different chromophores is particularly useful in assays where it is important to analyze substrate specificity. Accordingly, a wide variety of attached chromophores that absorb and emit at different wavelengths may be used. However, for less processive polymerases, an increased sensitivity for detection is advantageous. The coupling of fluorescent analogs to the nucleoside triphosphates increases the sensitivity of detection several-fold. For example, the fluorescent umbelliferone-GTP, is synthesized using the same one-step protocol for the synthesis of PNP-NTPs described herein. The umbelliferone serves as a nucleophile to modify the xcex3-phosphate. After the polymerase releases umbelliferone-pyrophosphate, alkaline phosphatase removes the phosphates, causing umbelliferone to fluoresce a bright blue color in the presence of UV light.
The invention provides kits for assaying polymerase reactions in standard laboratory spectrophotometers. The kits are designed so that the researcher can replace one or more components with the sample they wish to test. An exemplary kit contains the following components, all supplied at 10xc3x97the final concentration:
polymerase substrates including the colorimetric analog;
reaction buffer;
a nucleic acid template suitable for the polymerase;
a nucleic acid template not capable of directing polynucleotide synthesis, a negative control;
polymerase for positive control;
alkaline phosphatase; and
a reaction termination mix.
Exemplary label functionalities shown to be effective in the subject methods are shown in Table 1A; exemplary functionalized NTPs are shown in Table 1B.
In other embodiments of the invention, the functional group is a predetermined cell delivery enhancing and/or polymerase specificity enhancing moiety, xe2x80x94OR, wherein R is independently selected from substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl, each inclusive of carbocyclic and heterocyclic. These embodiments relate to nucleotide analogs that provide improved bioavailability and/or cellular uptake (e.g. through enhanced gut or blood stability, cellular and/or membrane permeability, host phosphatase stability, etc.) and/or selectivity, which are extremely important in nucleoside-based therapies. In a particular embodiment, the NTP provides specificity for a pathogenic polymerase, for example, overactive endogenous polymerases of neoproliferative cells and pathogenic bacterial, fungal and viral polymerases. Exemplary targetable bacterial polymerase sources include staphylococcus, exemplary fungal polymerase sources include candida; and exemplary viral polymerase sources include hepatitis viruses including HBV, HAV and HCV, rhinoviruses, influenza, hemorrhagic fever virus, HIV, etc.
For example, nucleoside analogs including AZT and acyclovir are among the most effective antiviral agents in current clinical use. Incorporation of nucleosides lacking a 3xe2x80x2 hydroxyl group into a viral nucleic acid prevent additional cycles of nucleotide addition and thereby inhibit the infection process. However, there are two significant drawbacks with this methodology: first, to function as a substrate for a polymerase, a nucleoside must be converted to a triphosphate. Because triphosphates are inherently unstable in an intercellular environment, they cannot be directly administered. Instead, the nucleoside is used as a prodrug, which is converted to a triphosphate via the familiar kinase-catalyzed steps shown below. Thus, for a compound to function as a drug, it must serve as a substrate in three separate phosphorylation steps in addition to serving as a substrate for the targeted polymerase. This is a relatively inefficient process, requiring antiviral nucleosides to be administered in high concentrations. Second, for an antiviral nucleoside to be effective, it must be a substrate for the target polymerase (and the kinases necessary to convert the molecule to a triphosphate), but not inhibit or deactivate cellular processes. Since many host enzymes use nucleoside triphosphates for essential biochemical processes, it has proven difficult to avoid host toxicity. Similar problems occur in many antifungal therapies. For example, flucytosine (5-fluorocytosine) is deaminated to 5-fluorouracil by pathogenic fungi, which can be subsequently metabolized to 5-fluorouridine triphosphate and incorporated into nascent RNA.
Our invention provides new strategies to overcome these problems associated with using nucleosides as therapeutic agents. We have found that attachment to the xcex3-phosphate of a nucleoside triphosphate of moieties which enhance cell delivery need not preclude its ability to function as a polymerase substrate. Furthermore, such modifications can provide the nucleoside triphosphate relatively enhanced stability toward degradation in serum and provide improved cellular uptake thus improving the potency of known antiviral nucleoside therapeutics by eliminating the need for these compounds to function as kinase substrates. Additionally, antiviral nucleosides that fail in-vivo because they are poor kinase substrates may become therapeutically effective when delivered as modified triphosphates. Furthermore, by increasing pathogen polymerase specificity, we can reduce the toxicity of these molecules toward the host.
Suitable cell delivery enhancing or polymerase specificity enhancing moieties are readily identified empirically. In particular, we generated libraries of modified nucleosides to screen in high throughput for polymerase-specific and cell delivery enhancing modifications. For example, in a particular screen, we use the one step synthesis described herein adapted to standard solid phase methods for parallel array synthesis, to generate moderate sized libraries of 1,000 modified triphosphates. In these syntheses, the resin bound triphosphates are the same, the only variable being the added alcohol. The nucleoside-resin linkage is adapted to the particular base, e.g. resin linkage via the amino functionality of the base in the case of guanine, and the imino function in the case of thymine.
Initial screens concentrated on two modified nucleosides as shown below. 
The triphosphate libraries are derived from AZT and dideoxyinosine (ddI, Bristol-Meyers Squibb, New York, N.Y.). These are tested against a panel of retroviral polymerases including eleven different HIV reverse transcriptase variants, as well as against human DNA polymerase. Compounds with highest activity against one or more viral targets and lowest activity against the human polymerase are selected for cell-based assays, such as cell penetration screens. To improve cellular penetration, the triphosphate group may be masked as the mixed acetal, as shown below. These uncharged molecules provide enhanced diffusion into cells, where they are subsequently unmasked by endogenous cellular esterases. 
Cell permeant, viral specific compounds may be subsequently modified with a colormnetric or fluorimetric label as described above for reverse transcriptase activity assays.
Cell delivery enhancing moieties encompass a wide variety of generally membrane permeant, relatively hydrophobic R substituents. Exemplary substituted or unsubstituted (C1-C18) alkyl, inclusive of carbocyclic and heterocyclic, cell delivery enhancing functionalities shown to be effective in the subject methods are shown in Table 2A; exemplary functionalized alkyl NTPs are shown in Table 2B. Exemplary substituted or unsubstituted (C1-C18) alkenyl, inclusive of carbocyclic and heterocyclic, cell delivery enhancing functionalities shown to be effective in the subject methods are shown in Table 3A; exemplary functionalized alkenyl NTPs are shown in Table 3B. Exemplary substituted or unsubstituted (C1-C18) alkynyl, inclusive of carbocyclic and heterocyclic, cell delivery enhancing functionalities shown to be effective in the subject methods are shown in Table 5A; exemplary functionalized alkynyl NTPs are shown in Table 4B. Exemplary substituted or unsubstituted (C1-C18) aryl, inclusive of carbocyclic and heterocyclic, cell delivery enhancing functionalities shown to be effective in the subject methods are shown in Table 5A; exemplary functionalized aryl NTPs are shown in Table 5B.
Polymerase specificity enhancing moieties are readily identified in comparative and competitive enzyme assays. Exemplary substituted or unsubstituted (C1-C18) alkyl, inclusive of carbocyclic and heterocyclic, polymerase specificity enhancing functionalities shown to be effective in the subject methods are shown in Table 6A; exemplary functionalized alkyl NTPs are shown in Table 6B. Exemplary substituted or unsubstituted (C1-C18) alkenyl, inclusive of carbocyclic and heterocyclic, polymerase specificity enhancing functionalities shown to be effective in the subject methods are shown in Table 7A; exemplary functionalized alkenyl NTPs are shown in Table 7B. Exemplary substituted or unsubstituted (C1-C18) alkynyl, inclusive of carbocyclic and heterocyclic, polymerase specificity enhancing functionalities shown to be effective in the subject methods are shown in Table 8A; exemplary functionalized alkynyl NTPs are shown in Table 8B. Exemplary substituted or unsubstituted (C1-C18) aryl, inclusive of carbocyclic and heterocyclic, polymerase specificity enhancing functionalities shown to be effective in the subject methods are shown in Table 9A; exemplary functionalized aryl NTPs are shown in Table 9B.
In the foregoing and other embodiments of the invention, the functional group may be or also comprise a moiety which upon polymerization of the nucleotide, provides a reaction product functionality such as a therapeutic or protherapeutic. A wide variety of bioactive molecules can be coupled to the nucleotide through the xcex3-phosphoester linkage. After polymerization, the pyrophosphate-linked functionality may be an active form, or may be further hydrolyzed to yield a bioactive or therapeutic molecule. Accordingly, this embodiment may further comprise a subsequent incubation in the presence of one or more phosphatases under conditions wherein the xcex2-xcex3 bond and/or xcex3-phosphoester bond is cleaved such that the liberated pyrophosphate-linked functionality is converted to the monophosphate and/or the unphosphorylated functionality. In addition, the same moiety may provide a plurality of functionalities, e.g. both cell delivery enhancing and prodrug functionalities. These functional groups provide particular application in targeting pathogenic polymerases, particularly pathogenic viral polymerases, and encompass a wide variety of hydroxyl-bearing substituents, xe2x80x94OR, wherein R is independently selected from substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl, each inclusive of carbocyclic and heterocyclic. Suitable moieties may be identified in viremia assays, such as described below, and/or derive from established therapeutics. For example, targetable functionalities include xcex3-phosphesters of a number of antiviral agents such as amantadine and rimantadine, xcex1, xcex2 and xcex3-interferons, nonnucleoside reverse transcriptase inhibitors such as nevirapine, delaviridine, loviride, etc. and protease inhibitors such as saquinavir, saquinavir mesylate, ritonavir, indinavir, nelfinavir, amprenavir etc. Coupling chemistry is readily selected by those of ordinary skill in accordance with the structure of the targeted functionality. For example, the foregoing peptide mimetic protease inhibitors all provide a single reactive hydroxyl group which is readily coupled to nucleoside triphosphates as outlined below. Exemplary pyrophosphate-prodrug liberating antiviral nucleotides made by these methods are shown in Table 10.
General synthetic scheme: Modifying a nucleoside triphosphate on the terminal phosphoryl group is technically straightforward. This is accomplished by treating the triphosphate tetramethylammonium carbonate with dicyclohexyl carbodiimide (DCC) in DMF to generate the cyclic anhydride (which is not isolated), followed by treatment with a nucleophile (ROH). After the reaction has proceeded for about 20 hours the solvent is evaporated and the solid residue purified to obtain the R-NTP. This strategy is diagramed below. 
Synthetic scheme for PNP-NTPs: ATP is treated with DCC to generate the cyclic anhydride; and then treated with a nucleophile 4-nitrophenol to give PNP-ATP. GTP is treated with dicyclohexyl carbodiimide (DCC) to generate the cyclic anhydride; and then treated with a nucleophile 4-nitrophenol PNP-GTP. CTP is treated with DCC to generate the cyclic anhydride; and then treated with a nucleophile 4-nitrophenol PNP-CTP. UTP is treated with dicyclohexyl carbodiimide (DCC) to generate the cyclic anhydride; and then treated with a nucleophile 4-nitrophenol PNP-UTP.